The glutathione transferases catalyze the nucleophilic addition of the sulfur of glutathione (GSH) to a wide variety of endogenous and xenobiotic substrates bearing electrophilic functional groups. They are arguably the single most important enzyme in the metabolism and detoxication of alkylating agents in mammals. In addition, they play diverse catalytic roles in the catabolism of foreign molecules in bacteria. It is the thesis of this proposal hat a thorough understanding of the participation of this group of enzymes in the metabolism of drugs and xenobiotics in both prokaryotes and eukaryotes must be based on a detailed knowledge of the relationship between molecular structure and catalytic mechanism. The investigations will focus on poorly understood aspects of the structure and catalytic mechanism of selected mammalian and bacterial enzymes. Four specific aims will be pursued. First, the recently discovered mammalian mitochondrial (class kappa) enzyme, which appears to be more closely related to a bacterial isomerase than to other mammalian GSH transferases, will be investigated by: (i) determination of its three dimensional structure; (ii) the definition of the interaction of the enzyme with GSH by preequilibrium kinetics and mutagenesis; and (iii) isolation and mechanistic analysis of the orthologous 2-hydroxychromene-2- carboxylate isomerase in the naphthalene catabolic pathway of Pseudomonas putida. Second, a unique dichloromethane dehalogenase from Methylophylis sp. which appears to catalyze the hydrolytic dechlorination of dihalomethanes without releasing mutagenic S-halomethyl glutathione intermediates during turnover will be investigated. The mechanistic basis for this phenomenon will be established by: (i) steady-state and presteady-state kinetic analysis of the dehalogenation reaction; (ii) a determination of the chemical stability of intermediates in the reaction; (iii) mutagenic analysis of suspected active site residues; and (iv) crystallization and determination of the three-dimensional structure of the enzyme. Third, dimer interface mutants of mammalian class mu enzymes will be studied in an effort to understand the role of the interface in the structural integrity and catalytic activity of individual subunits. These investigations will include: (i) a full mechanistic analysis of mutants at key positions (F56 and D105) identified by X-ray crystallography; (ii) determination of the effects of the mutations on the kinetics of unfolding of the enzyme; and (iii) crystallization and structure determination of the dimer interface mutants. Finally, the enigmatic mechanism of the microsomal enzyme will be explored by: (i) probing the interaction of the enzyme with GSH by preequilibrium kinetic techniques; (ii) mutagenic analysis; and (iii) X-ray crystallography.